Cryopreservation of bacteria
with special reference to anaerobes
Khursheed A. Malik, Ph.D.
DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Mascheroder Weg 1b, D-3300 Braunschweig, Federal Republic of Germany
World Federation for Culture Collections
Technical information sheet No 4
Published by: UNESC/WFCC - Education Committee 1989
Introduction
The methods used in the maintenance of stock cultures of microorganisms usually
involve serial subculturing or other simple methods of preservation which are
not only time consuming but can also result in genetic instability.
Cryopreservation of microorganisms in liquid nitrogen at -196C is a very
reliable method and is generally considered superior to other preservation
methods. Bacteria preserved in liquid nitrogen normally show high survival rates
and good strain stability during long-term storage. In liquid-nitrogen storage
of microorganisms polypropylene cryotubes, glass vials, glass capillaries and
polypropylene straws are generally used. In the method described here, screw-cap
polypropylene cryotubes and mini-screw cap glass ampoules have been used. The
latter are also suitable for the preservation of bacteria under anaerobic
conditions. The method is simple, effective and economical with respect to the
storage space and costs. In the preservation of anaerobes, generally no
continuous stream of nitrogen gas, no anaerobic chamber or glove boxes are
required. This method provides a suitable model for cryogenic storage of many
fastidious and delicate bacteria.
General considerations for successful cryopreservation
Several factors can affect cell viability and stability during cryopreservation.
During cryopreservation, dehydration of cells results and osmotic imbalance is
created due to the changes in the concentration of salts and other metabolites.
During the cooling process rupture of the cellular membranes can also occur by
the formation of large ice crystals. Successful preservation can be achieved by
the use of cryo-protective agents (such as dimethylsulfoxide, glycerol),
maintaining a controlled rate of cooling (about 1C per minute to about -30'C)
and an appropriate rewarming protocol (rapid thawing in a 37C water bath which
takes about one minute for a glass ampoule and somewhat longer for a plastic
vial ). In practice, a relatively slow cooling rate can be easily obtained by
keeping ampoules/vials in mechanical deep-freezers for 1-2 hours or in the neck
of the liquid nitrogen storage unit for some minutes and then lowering
containers into it. It is, however, not good practice to plunge cultures
directly into liquid nitrogen, as the liquid nitrogen may seep into any
imperfectly closed or sealed capillaries, ampoules, or vials containing the
bacterial suspensions. On removal from storage, nitrogen (inside an ampoule)
will virtually instantly change to the gaseous phase causing an explosion. For
safety reasons it is thus recommended that cultures should be stored in the gas
phase of liquid nitrogen.
While preparing cells for cryopreservation several factors such as optimal
growth conditions, physiological state of the cells (preferably from the late
logarithmic to early stationary phase of growth), high cells density (106 to 10
cells per ml) should be considered as these can affect cell viability after
cryopreservation. After mixing, cell suspensions should be kept for
equilibration with the cryoprotective agent. For harvesting, liquid cultures are
centrifuged. However, vigorous pipetting and high-speed centrifugation should be
avoided and cells should be handled gently. Viability assays should be performed
on all cultures before and after cryopreservation to assure long-term viability.
To assure purity, identity of the preserved cultures should be verified and
after freezing cultures should be recharacterized to assure their stability.
Safety precautions should be observed when removing an ampoule from liquid
nitrogen. The face shield, laboratory coat and insulated gloves should be worn
as protection against liquid nitrogen splash and exploding ampoules. The level
of the liquid nitrogen in the containers should be checked preferably on a daily
basis and maintained to a constant level, as any drop in liquid nitrogen level
below a critical volume can result in damage due to the warming of the samples.
Materials and Methods
Equipment
Screw-capped plastic cryovials of about 2 ml capacity (available from Nunc,
Gibco Europe Ltd., or Nalge Company). These are supplied in sterile packings.
These are not suitable for repeated use. Screw-cap glass ampoules (10x30 mm) of
2 ml capacity (available from Varian GmbH, Darmstadt FRG). These are generally
used for autosamplers in gas chromatography and are provided with rubber septa
and plastic screwcaps with holes for the injection of the samples. It is
recommended to use ampoules with black screw-caps and oxygen-impermeable butyl
rubber septa. The ampoules are washed, rinsed with distilled water, tightly
closed and autoclaved. Before use these are labelled with the numbers of the
strains to be preserved. Liquid nitrogen storage tanks with canisters, racks,
canes, (supplied by Union Carbide; Air Liquide, France; Messer Griessheim, FRG;
or else where) and precautionary arrangements like safety glasses, gloves etc.
Anaerobic facilities for the preparation of reduced media. Hungate tubes with
septa (Bellco Glass Inc., 2047-16125). Butyl rubber overflow tubes (about 5 mm
diameter) with Luer Lock adapters at both ends and long syringe needles (10-15
cm in length). Sterile gas tight hypodermic Luer Lock syringes. Cryoprotective
agent glycerol and dimethylsulfoxide (DMSO), reagent grade. Glycerol (20% w/v in
H20) may be sterilized by autoclaving for 15 minutes and is stored in screw-cap
bottles in dark. DMSO (20% v/v in H20) is sterilized by filtration (using a
Teflon syringe filter) or can be autoclaved undiluted at 114C for 10 minutes.
Preparation of cell suspension for freezing
The aerobic cultures to be frozen are grown in appropriate media (under well
aerated conditions ) and should be harvested preferably in active phase of
growth. From agar slants the cultures are removed with a loop and gently
suspended in sterile glycerol (10% w/v) or DMSO (5% v/v) to obtain a heavy cell
suspension. For anaerobes this is done under a stream of sterile nitrogen gas.
Thick suspensions (108 -10'' cells/ml) of liquid cultures are mixed in equal
quantities with the double concentrated cryoprotective agents. For harvesting,
the anaerobic cultures are centrifuged for 30 minutes at 4000 xg in the
screw-cap bottles in which cultures are grown. The supernatant is removed
anaerobically under a stream of nitrogen gas using an overflow butyl rubber tube
of about 5 mm diameter with Luer Lock adapters at both ends and fitted with long
syringe needles of 1015 cm length (see Fig. 1A). To obtain sterile nitrogen gas
a sterile, cotton filled syringe is attached to a conduit connected to the N2
gas (99.99%) cylinder. The pellet is resuspended carefully in ice cold sterile
DMSO solution (5% v/v in H2O). In the case of halophilic strains or cells which
do not form a pellet a thick bacterial suspension (in growth medium) is mixed in
the ratio 3:1 with ice cold sterile DMSO (20% v/v in H2O). For extreme
halophilic strains optimum salt concentration should be maintained after mixing
cell suspension with the DMSO. The cells are allowed to equilibrate with the
cryoprotectant (15 minutes for DMSO, 30 minutes for glycerol) in an ice bath.
Filling of ampoules and freezing
While equilibrating, an aliquot of 1.0 to 1.5 ml of cell suspension is dispensed
in to each plastic cryovial or glass ampoule. For anaerobes using a sterile
gas-tight 5-10 ml syringe, the ampoules are evacuated for anaerobiosis and to
facilitate filling (Fig. 1B). About 1 ml of thick cell suspension (equilibrated
with the DMSO) is withdrawn with a 1 ml sterile oxygen free syringe (already
flushed with nitrogen gas) and injected into each ampoule (Fig. 1C) Immediately
after the glass ampoules or cryotubes are clamped onto labeled aluminium canes,
placed at -30C for about one hour or for few minutes in the gas phase of liquid
nitrogen. The canes are then placed in canisters, racks or drawers and frozen by
direct immersion in liquid nitrogen or in the gas phase of liquid nitrogen (Fig.
1D).
Revival of cultures
The frozen ampoule is removed from liquid nitrogen. For partial thawing these
are immediately immersed to the neck in the mini water bath at 37C (Fig. 1E) for
a few seconds. After thawing the outer surface of the ampoules is dried by
wiping and plastic vials are wiped with alcohol-soaked gauze prior to opening.
For aerobic bacteria the screwcap glass vials can be opened and flame sterilized
at the neck. The thawed contents of the ampoule/vial are immediately transferred
to fresh growth medium to dilute the cryoprotectant, which other wise is lethal
at higher temperatures. For anaerobes the septum of the glass ampoule is flame
sterilized after putting a drop of alcohol and with a 1 ml oxygen free syringe a
small volume (about 0.05 ml) of inoculum is withdrawn and injected into 5-10 ml
liquid growth medium (Fig. 1F). The rest of the cell suspension is immediately
frozen again (a self made wax block rack, chilled to -30C is used for
transportation to the liquid nitrogen container see Fig. 1G) in liquid nitrogen
for later use. In this way one ampoule can be used for several repeated
retrievals or inoculations. The DMSO which is often toxic during growth is
diluted 100-200 times in the culture medium to a non inhibitory concentration.
The inoculated growth medium is incubated under appropriate growth conditions.
Estimation of viability counts
For aerobic bacteria, 0.5 ml of inocula is transferred to 4.5 ml of liquid
growth medium and serial decimal dilutions are prepared. Plating and counting
are done using standard methods. For the estimation of viable cell counts in
anaerobic bacteria, 0.5 ml of inocula is transferred from the unfrozen (for cell
counts before freezing) and from the thawed cell suspension (for cell counts
after freezing) into prereduced 4.5 ml medium in screw-cap tubes (Hungate tubes
with septa, Bellco Glass Inc., 2047-16125) and 6-8 serial decimal dilutions are
prepared using oxygen free syringes and incubation is done under appropriate
conditions. Agar roll tubes can be prepared for viable colony counts
determination if such facilities are available. Colony counts on agar plates can
be performed in an anaerobic glove box or anaerobic jars. Single plates can be
incubated anaerobically in anaerobic Biobags (Type A, Marion Scientific
Corporation, Kansas City, Ml, USA) or in Anaerocult bags (E. Merck, Postfach
4119, D-6100 Darmstadt). In the case of viable colony counts in agar roll tubes
or on plates the number of colonies are counted from each dilution and average
colony forming cells per sample are calculated.
For cultures which are difficult to grow in, or on agar, only liquid dilutions
series are made. In this case the number of cells is determined using the most
probable number method (MPN).
For comparison the viable cell counts before freezing and after freezing are
recorded and percentage survival is calculated.
Selected references for further reading
Greiff,D., H. Melton and T.W. Rowe, 1975. On the sealing of gas-filled glass
ampoules. Cryobiology 12: 1-14.
Kirsop, B. and J.J.S. Snell (eds.), 1984. Maintenance of Microorganisms: A
Manual of Laboratory Methods. Academic Press, London.
Malik, K.A., 1977. Rapid surface colony counts determination with three new
miniaturised techniques. Zbl. Bak. Hyg. I. Abt. A. 237: 415-423.
Malik, K.A., 1984 . A new method for liquid nitrogen storage of phototrophic
bacteria under anaerobic conditions. Journal of Microbial Methods 2: 41-47
Malik, K.A., 1985. Modern Methods of Gene Conservation. A Laboratory Manual.
PASTIC Press,lslamabad, Pakistan.
Simione, F.P., P.M. Daggett, M.S. MacGrath and M.T. Alexander, 1977. The use of
plastic ampoules for freeze preservation of microorganisms. Cryobiology 14:
500-502